The objective of this proposal is to demonstrate that naturally occurring photoproteins and their mutants can be used in the design and development of highly sensitive luminescent assays capable of small volume analysis. In particular, we propose to prepare and characterize new genetically engineered variants of aequorin, obelin, and other photoproteins, so that they are capable of emission of light at different wavelengths than those of the corresponding wild-type photoproteins. These photoproteins will be then employed as reagents in the development of assays for physiologically important molecules and for the high-throughput screening of biopharmaceuticals. By using the different variants of the photoproteins, array detection of biomolecules will be investigated. Research performed by our group during the current budget period demonstrated the usefulness of employing recombinant conjugates in the highly sensitive detection of biomolecules. We now propose to design and develop new methods based on the bioluminescence of photoproteins that are suitable for the microanalysis of important biomolecules, such as vitamins, drugs, peptides, etc., in nL-sized samples.